in vivo 26: 665-670 (2012)
Chrysin, a Natural and Biologically Active Flavonoid, Influences a Murine Leukemia Model In Vivo through Enhancing Populations of T-and B-Cells, and Promoting Macrophage Phagocytosis and NK Cell Cytotoxicity CHIN-CHUNG LIN1,2, CHUN-SHU YU3, JAI-SING YANG4, CHI-CHENG LU5, JO-HUA CHIANG5, JING-PIN LIN6, CHAO-LIN KUO7 and JING-GUNG CHUNG8,9 1Department
of Chinese Medicine, Fong-Yuan Hospital, Department of Health, Executive Yuan, Taichung, Taiwan, R.O.C.; 2School of Medicine and Nursing, Hunkuang University, Taichung, Taiwan, R.O.C.; Schools of 3Pharmacy, 6Chinese Medicine, and 7Chinese Pharmaceutical Sciences and Chinese Medicine Resources, Departments of 4Pharmacology and 8Biological Science and Technology, China Medical University, Taichung, Taiwan, R.O.C.; 5Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, R.O.C.; 9Department of Biotechnology, Asia University, Taichung, Taiwan, R.O.C.
Abstract. Chrysin (5,7-dihydroxyflavone), a natural and biologically active flavonoid found in plants, possesses many biological activities and anticancer effects. However, there is no available evidence regarding the antileukemia responses to chrysin in a mouse model. We hypothesized that chrysin affects murine WEHI-3 leukemia cells in vitro and in vivo. The present study showed that chrysin at concentrations of 5-50 μM reduced the cell viability in concentration- and time-dependent manners. In an in vivo study, WEHI-3 leukemic BALB/c mice were established in order to determine antileukemia activity of chrysin. Our results revealed that chrysin increased the percentage of CD3 (Tcell maker), CD19 (B-cell maker) and Mac-3 (macrophages) cell surface markers in treated mice as compared with the untreated leukemia group. However, chrysin did not significantly influence the level of CD11b (a monocyte maker) in treated mice. Moreover, there was a significant increase in phagocytosis by macrophages from peripheral
Correspondence to: Jing-Gung Chung, Ph.D., Department of Biological Science and Technology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan, R.O.C. Tel: +886 422053366 ext. 2161, Fax: +886 422053764, e-mail:
[email protected] and ChaoLin Kuo, Ph.D., School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University. No 91, HsuehShih Road, Taichung 40402, Taiwan. Tel: +886 422053366 ext. 5202, Fax: +886 422070439, e-mail:
[email protected] Key Words: Chrysin, WEHI-3 murine leukemia cells, T- and B- cells, macrophage phagocytosis, NK cell cytotoxicity, CD3, CD19, CD11b.
0258-851X/2012 $2.00+.40
blood mononuclear cells, but no effect in those from the peritoneal cavity in leukemic mice after chrysin treatment. Isolated splenocytes from chrysin-treated leukemic mice demonstrated an increase of natural killer (NK) cell cytotoxicity. Based on these observations, chrysin might exhibit antileukemia effects on a murine WEHI-3 cell lineinduced leukemia in vivo. In Taiwan, based on a 2010 report from the Department of Health, Executive Yuan, R.O.C. (Taiwan) 4.2 individuals per 100,000 thousand die from leukemia. Currently, the treatments for leukemia are radiotherapy, chemotherapy, or a combination of radiotherapy with chemotherapy; however, these treatments have proven unsatisfactory. Thus, many investigators have focused on new compounds for the treatment of leukemia patients. Numerous studies have been demonstrated on the track that an increased consumption of a plant-based diet can reduce the risk of cancer development (1-3). Therefore, new compounds from natural plant are the major focus for investigators because diet can play a vital role in cancer prevention (4). Chrysin (5,7-dihydroxyflavone) is a naturally active compound of the flavone group and can be obtained from honey, propolis and plants (4-6). Chrysin has been reported to exert multiple biological activities such as antiinflammatory (7) and anti-oxidation effects (8), and cancer chemopreventive activity through inducing cell cycle arrest (5, 9) and apoptosis in melanoma (5) and leukemia cells (9). Chrysin reduced melanoma cell proliferation and induced cell differentiation in both A375 human and B16-F1 murine
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in vivo 26: 665-670 (2012) melanoma cells (10). Interestingly, chrysin has been shown to induce apoptosis in U937 (9, 11), HL-60 and L1210 leukemia cells (12). Furthermore, chrysin has been shown to inhibit tumor angiogenesis in vivo and angiogenesis is a critical step in cancer cell metastasis (13). Currently there are no reports to address the effects of chrysin on the immune system of leukemic mice in vivo. Thus, in the present study, we investigated whether chrysin can promote phagocytosis of macrophages and increase activity of natural killer NK cells from leukemic BALB/c mice in vivo.
Materials and Methods Materials and reagents. Chyrsin, dimethyl sulfoxide (DMSO), propidium iodide (PI), RNase A and Triton X-100 were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). RPMI-1640 medium, fetal bovine serum (FBS), L-glutamine and penicillinstreptomycin were purchased from Gibco Life Technologies (Carlsbad, CA, USA). WEHI-3 murine leukemia cells. The WEHI-3 murine myelomonocytic leukemia cell line was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan, ROC). Cells were immediately maintained in plastic culture flasks (75 cm2) in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin at 37˚C under a humidified atmosphere with 5% CO2. The cells were cultivated for two complete cycles in an incubator. Viability determination. About 2×105 WEHI-3 cells/well were placed into 24-well plates for 24 h. Chrysin was dissolved in DMSO then was individually added to the wells at final concentrations of 0, 5, 10, 20, 30, 40 and 50 μM, and 0.1% of DMSO in culuture medium was added to the well as the control group. After treatments for 24 and 48 h, cells from each well were harvested for the determination of viability by using a flow cytometric method as described previously (14, 15). Male BALB/c mice. Thirty male BALB/c mice of 8 weeks of age and around 22-25 g in weight were purchased from the Laboratory Animal Center, College of Medicine, National Taiwan University (Taipei, Taiwan, ROC). This study following the institutional guidelines (Affidavit of Approval of Animal Use Protocol) was approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University (Taichung, Taiwan). Establishment of leukemic mice and chrysin treatment. Thirty BALB/c mice were intraperitoneally (i.p.) individually injected with 1×105 WEHI-3 cells for 2 weeks and then were randomly separated into three groups as a model of leukemia. Group I mice were treated with olive oil (vehicle) as control (10 animals). Group II mice were treated with chrysin (10 mg/kg) in olive oil (10 animals). Group III animals were treated with chrysin (50 mg/kg) in olive oil (10 animals). Chrysin was administered by oral gavage to the treatment groups at the above doses daily for 2 weeks before mice were weighed and sacrificed by euthanasia with CO2 (16). Quantification of phagocytic activity of macrophages. The measurement of phagocytosis in each animal was performed by the
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Figure 1. Chyrsin reduced the percentage of viable WEHI-3 cells. Cells were treated with 0, 5, 10, 20, 30, 40 and 50 μM chrysin for 24 and 48 h. The cells were harvested and the percentages of viable WEHI-3 cells were determined as described in Materials and Methods. Each point is the mean±S.D. of three experiments. *p
